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  • Title: High-level synthesis of recombinant HIV-1 protease and the recovery of active enzyme from inclusion bodies.
    Author: Cheng YS, McGowan MH, Kettner CA, Schloss JV, Erickson-Viitanen S, Yin FH.
    Journal: Gene; 1990 Mar 15; 87(2):243-8. PubMed ID: 2158928.
    Abstract:
    A complete chemical synthesis and assembly of genes for the production of human immunodeficiency virus type-I protease (HIV-PR) and its precursors are described. The T7 expression system was used to produce high levels of active HIV-PR and its precursors in Escherichia coli inclusion bodies. The gene encoding the open reading frames of HIV-PR was expressed in E. coli as a 10-kDa protein, while the genes encoding HIV-PR precursors were expressed as larger proteins, which were partially processed in E. coli to the 10-kDa form. These processing events are autoproteolytic, since a single-base mutation, changing the active-site aspartic acid to glycine, completely abolished the conversion. HIV-PR can be released with 8 M urea from washed cellular inclusion bodies, resulting in a preparation with few bacterial host proteins. After refolding, this preparation contains no nonspecific protease or peptidase activities. The recombinant HIV-PR isolated from inclusion bodies cleaves HIV-PR substrates specifically with a specific activity comparable to column-purified HIV-PR.
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